Enrichment of DNA fragments for deleted sequences in human genome with nuclease S1 digestion and PCR amplification.

Recently, a system combining subtractive and kinetic enrichment of DNA fragments present in one complex genome but not in its normal or deviant counterpart has been developed (1,2,3)The authors designate this strategy 'difference cloning' and describe their protocols in detail (1,3). During our long-term endeavor for identifying disease genes for esophageal cancer, a common malignancy in North China, various strategies have been explored, among which a procedure similar to that proposed by Lisitsyn, et al. (3) but with some modifications turned out to be effective in this connection. We describe here our protocol and results obtained. For the sake of simplicity in description, we adopt the terms suggested by Wigler's group in their original meanings, e.g., 'driver' means a genome or DNA population lacking the 'target', and 'tester' the same genome or DNA population harboring the 'target', etc. To test the feasibility of our method we also used placenta DNA as driver and the same placenta DNA with equimolar X DNA as tester. The tester DNA was digested with Nspl endonuclease and ligated to Nspl adaptors (5'-CGGGAATTCGCTCGACATG-3'). The ligation reaction was carried out with 3'-CATG of the adaptor complemented to Nspl digested DNA ends, Klenow enzyme filled the protrudings formed following the adaptor complementation in the presence of four deoxynucleotides, and T4 ligase ligated the nick between the Nspl ends and adaptors. The driver was prepared from placenta DNA sonicated to a size range of 0.5 -1 .0 kilobase pairs. The tester and driver were mixed at a ratio of 1:200 to a final concentration of 10 mg/ml. The denatured mixture was then reannealed for 20 hours at 68°C in a total volume of 5 /il containing 1.0 M NaCl and 30 mM Tricine (Serva) buffer (pH 8.3). After the reassociation, die mixture was diluted five folds with sterile water, and the DNA was then precipitated by addition of three volumes of ethanol. The precipitate was dissolved in 100 nl of 200 mM NaCl, 50 mM sodium acetate (pH 5.0), 2 mM zinc chloride, and 6000 u nuclease SI (100 units//tg of DNA) and incubated at 16°C for 50 minutes. The resultant material was precipitated with ethanol, and redissolved in 5 /il of 36 mM Tricine (pH 8.3). Four fifths of the solution was used to repeat the denatured DNA reassociation and SI digestion according to the above step and one-fifth of the solution to undertake PCR amplification. The process of hybridization in solution and nuclease SI treatment was reiterated three times. Figure 1 shows the results of the PCR amplification of DNA resulted from first, second and third round subtraction. At the third round of the reaction some discriminated fragments were visible. These enriched fragments were about the sizes of Nspl digested X DNA. Southern blot hybridization not only proved that these fragments were originated from X DNA but showed that the enriched PCR products retained the most of the X DNA fragments (Fig. 2). The major advantages and modifications of this SI digestion method are: (i) making use of SI digestion to remove all of the adaptors in DNA duplexes formed between tester and driver, and to destroy the mismatched sequences but to retain the exactly reannealed target sequences flanked with intact adaptors as primer